Restriction amplification assay

ABSTRACT

The present invention relates to a method, reagent and kit for the determination of the presence of target nucleotide sequences by restriction amplification. In the process to detect nucleic acid sequences a target molecule containing a specific restriction site is hybridized with a labeled probe containing a sequence homologous to at least 28 bases of the target molecule. The probe is cleaved with a restriction enzyme that releases the probe for detection if the probe hybridizes to the specific target. A second oligonucleotide is present in the reaction that is homologous to the 3 prime end of the probe molecule and also conains 5 prime base or bases that will reconstitute the restriction enzyme site on the target. Thus, the cleaved probe constantly regenerates and is highly detectable if the target sequence is present in the assay.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods, reagents and kits for thedetermination of the presence of target nucleotide sequences. Inparticular, the present invention relates to a method for conducting anassay for the presence of a specific target nucleotide sequence in asample containing an unknown quantity of said specific target nucleotidesequence and to probes for use in such an assay.

2. Description of the Prior Art

In the technology of manipulating genetic material or in evaluating thegenetic character of an organism, it is often desirable to ascertain ifa particular gene or part of a gene is present in an organism or in anextracellular extract of genetic material from that organism. Since anygene or gene portion is, in essence, a specific sequence of nucleotidebases forming all or part of a polynucleotide molecule, it is possibleto directly test the sample polynucleotide to discover if the specificsequence of nucleotide bases forming the gene is present in the sample.

Interest in specific sequences of nucleotide bases may involve detectingthe presence of pathogens, determining the presence of alleles,detecting the presence of lesions in a host genome and detecting aparticular mRNA or the modification of a cellular host, to name only afew illustrative examples. Genetic diseases such as Huntington chorea,muscular dystrophy, phenylketonuria, thalassemias and sickle cell anemiacan be diagnosed by the analysis of an individual's DNA. Furthermore,diagnosis or identification of viruses, viroids, bacteria, fungi,protozoa or any other plant or animal life form can be determined byhybridization assays with nucleotide probes.

Nucleic acid detection assays of various types have been documented inthe literature. These types of assays, and in particular those requiringdetection of polynucleotides, are based on the purine-pyrimidine basepairing properties of complementary nucleic acid strands in DNA-DNA orDNA-RNA duplexes This base-pairing process most frequently occursthrough formation of hydrogen bonds in the pairing of adenosine-thymine(A-T) and guanosine-cytosine (G-C) bases in double-stranded DNA;adenosine-uracil base pairs may additionally be formed by hydrogenbonding in DNA-RNA hybrid molecules. Base pairing of nucleic acidstrands for determination of the presence or absence of a givennucleotide sequence involving sample nucleotide sequences and a probenucleotide sequence is commonly referred to as nucleic acidhybridization or simply hybridization.

One of the most powerful tools of molecular biology is the ability tofractionate nucleic acids and to determine which nucleic acids havesequences complementary to an array of DNA or RNA molecules. TheSouthern blot is a well known method for transferringelectrophoretically fractioned DNA from a gel matrix to a nitrocellulosesolid support by passive diffusion, followed by hybridization to alabeled probe. Similar procedures are used for detecting RNA with minormodifications and this method is known in the art as the Northern blot.The use of dried agarose gels as the immobilized phase is known as theUnblot method. All of these assay techniques are valuable tools foranalyzing mRNA's, clones, genes, fragments, flanking sequences,repetitive elements and the like.

U.S. Pat. No. 4,358,535 describes a method for detecting pathogens usinga target nucleic acid sequence. The method involves fixing a targetnucleic acid sequence to an inert support before hybridization with aradioactively labeled nucleotide probe. The target nucleic acid sequenceis then determined by detecting the presence of any label on the inertsupport.

European Patent Application No. 0 117 440 discloses non-radioactivechemically labeled polynucleotide probes and methods of using theprobes. The target nucleic acid sequence is also fixed to a solidsupport.

U.S. Pat. Nos. 4,767,699 and 4,795,701 disclose a nucleic aciddisplacement assays. These assays utilize two polynucleotides; onepolynucleotide is labeled, and the other polynucleotide is used todisplace the labeled probe from the target sequence, thereby allowingdetection of the target molecule. These assays use ATP to detect whetherhybridization has occurred with the target molecule.

Many of the assays using nucleotide probes have problems in thedetection systems. Sensitivity of the labeled probe and backgroundlevels that are generated during the assay often lead to erroneousresults.

To facilitate more efficient detection of a nucleic acid sequence from agiven sequence of DNA or RNA a target amplification method may beutilized. This method, known as PCR, is described in U.S. Pat. No,4,683,195 and uses a set of primers and a DNA polymerase to extend thenucleic acid sequence of the target nucleotide and amplify it for futureprobe detection. By amplifying the DNA sequence, the target nucleotidecan be more efficiently detected with the nucleotide probe. One of theproblems encountered in this probe assay is contamination of thereaction medium.

Type II restriction enzymes are known in the art for makingdouble-stranded scissions at specific sites within a DNA molecule. Theseenzymes are prevalent in bacteria, contain only one type of subunit andMg²⁺ alone is required for DNA cleavage. DNA cleavage or scission occursat specific sites within or adjacent to the enzyme's recognition site.More than 400 restriction enzymes have been isolated from bacterialstrains to date. These restriction enzymes have been characterizedprimarily with respect to their recognition sequences and cleavagespecificity. The majority of restriction enzymes or endonucleasesrecognize sequences 4-6 nucleotides in length, but some have been foundwith 7-8 base recognition sites. Most, but not all, recognition sitescontain a dyad axis of symmetry and in most cases all the bases withinthe site are uniquely specified. Recognition of the symmetrical sequenceof the hybridized sequences or palindromes is made by endonucleases.Endonucleases with symmetrical recognition sites generally cleavesymmetrically.

The use of restriction enzymes with their specific cleavage sites iswell recognized in the art. Usually restriction enzymes are used for thespecific mapping, cloning and characterization of DNA sequences.However, they have been used in various nucleotide probe assays. Forinstance, U.S. Pat. No. 4,683,194 discloses a method for detecting thepresence or absence of a specific restriction site in a nucleic acidsequence by hybridization with a nucleic acid probe that iscomplementary to one strand of the nucleic acid sequence spanning therestriction site. The hybridized sequence is then cleaved with arestriction enzyme and the resulting cut and uncut oligomers areseparated and detected based on the type of probe label.

A similar concept for detecting a target nucleotide having ahalf-restriction site is set forth in U.S. Pat. No. 4,725,537. Thispatent discloses the use of a restriction endonuclease in adisplacement-type of assay.

Another type of assay that uses the concept of cleaving a nucleic acidsequence in a nucleotide probe is disclosed in U.S. Pat. No. 4,876,187.This method is used to detect DNA or RNA sequences by specificallycleaving the nucleic acid sequence of the probe at in least one pointthereby removing any reporter molecules not bound to a complementarytarget DNA sequence. This assay improves the signal to noise ratio ofthe detection system and is a highly sensitive assay.

Although the aforementioned assays do provide a method for detectingnucleic acid sequences in a target molecule, the need still exists foran assay system that provides very high sensitivity, ease of detection,less contamination in the assay medium and ease of operation, whileavoiding false positive results.

The present invention overcomes the disadvantages associated with thetechniques discussed above by introducing a highly sensitive detectionmethod for detecting a nucleic acid sequence through the use of a novelform of restriction amplification. A second oligonucleotide is presentin the assay to recycle the cleaved target sequence of interest therebyamplifying the labelled and cleaved probe oligonucleotide.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is provide a highlysensitive nucleic acid recycling or probe amplification assay fordetecting a nucleic acid sequence.

Another object of the present invention is to provide an assay thatrecycles the target sequence of interest and thereby amplifies thelabeled probe.

The present invention provides a method for detecting the presence of anucleic acid sequence in a biological sample, said method comprising thesteps of:

(a) providing a first oligonucleotide which comprises a nucleic acidsequence and a scissile linkage that is substantially complementary to anucleic acid target sequence, said first oligonucleotide having adetectable marker attached thereto;

(b) adding a second oligonucleotide to form a mixture that issubstantially complementary to an end of the first oligonucleotide thatdoes not rehybridize to the target sequence and comprising at least onebase that will reconstitute the cleaved scissile linkage on a targetmolecule;

(c) adding a cleaving enzyme to said reaction mixture which is able tocleave the scissile linkage if said target sequence and said firstoligonucleotide hybridize;

(d) hybridizing said reaction mixture; and

(e) detecting the cleaved detectable marker.

Another embodiment of the present invention recites:

A method of detecting in a sample the presence of a nucleic acidsequence, said method comprising the steps of:

(a) denaturing a target nucleic acid sequence containing a scissilelinkage in the presence of:

(i) a first oligonucleotide which contains a scissile linkage and issubstantially complementary to the nucleic acid sequence of the targetmolecule; (ii) a second oligonucleotide that is substantiallycomplementary to the 3 prime end of said first oligonucleotide andcomprises a 5 prime base or bases that will reconstitute the cleavedscissile linkage on said target molecule to form a mixture;

(b) adding a cleaving enzyme to said mixture;

(c) permitting said reaction mixture to hybridize whereby the cleavingenzyme will release the detectable marker from said firstoligonucleotide sequence; and

(d) detecting the cleaved detectable marker.

Still another embodiment of the present invention recites:

A method for detecting the presence of a nucleic acid sequence in abiological sample, said method comprising the steps of:

(a) hybridizing a target molecule having a scissile linkage to alabelled first oligonucleotide probe that has a complementary sequenceto said target and a detectable marker to provide a probe:target duplex;

(b) cleaving said duplex at the scissile linkage;

(c) adding a second oligonucleotide that reconstitutes the cleavedscissile linkage on the target molecule;

(d) recycling the reconstituted target molecule; and

(e) detecting said cleaved first oligonucleotide probe.

Yet another object of the presence invention is to provide kits fordiagnosis of various diseases using the above-described method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the method of the presentinvention with a complementary target sequence.

FIG. 2 is a schematic representation of the method of the presentinvention with a non-complementary non-target sequence.

FIG. 3 is an autoradiograph of several samples run using the presentassay.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

More particularly, the present invention relates to a method fordetecting nucleic acid sequences of interest in a target molecule. Thisassay uses a second oligonucleotide that is able to reconstitute thescissile linkage of said target molecule, permitting cleavage of thefirst oligonucleotide, thereby recycling the target sequence andamplifying the amount of the cleaved first oligonucleotide probe.

The term "oligonucleotide" as used herein refers to a compound made upof the condensation of a small number of nucleotides. The exact size ofthe oligonucleotide will depend on many factors including the ultimatefunction of the use of the oligonucleotide.

As used herein, the term "scissile linkage" refers to a site-specificrecognition nucleotide sequence that is cleavable by the use ofrestriction enzymes or restriction endonucleases.

As used herein, the terms "restriction endonucleases" and "restrictionenzymes" refer to bacterial enzymes each of which cut double-strandedDNA at or near a specific recognition nucleotide site.

The sample nucleic acid which may be employed herein may be derived fromany source(s), including organisms, provided that it contain theparticular restriction site of interest within a given nucleic acidsequence. Thus, the process may employ pure DNA which is single ordouble stranded or a cDNA or a mixture of nucleic acids, provided theycan be cut by restriction endonucleases. Sources include, for example,plasmids such as pBR322, cloned DNA, genomic DNA from any source.Typical sources can be from biological samples including bacteria,yeasts, viruses, and higher organisms such as plants, birds, reptilesand mammals.

Genomic DNA may be prepared from blood, urine, tissue material such aschorionic villi or amniotic cells by a variety of techniques (such asthose described by Maniatis et al., in Molecular Cloning, (1982)280-281). If necessary or desired to lower the viscosity, the sample ofprepared human DNA to be analyzed may be physically sheared or digestedusing a specific restriction endonuclease.

The first oligonucleotide used in the present invention is asingle-stranded oligonucleotide and has a structure complementary to thenucleic acid sequence being detected. The probe is usually DNA and maycontain an unlimited number of bases. However, it is preferable that theprobe contains up to about 100 bases, more preferably between about 10to 40 bases. The probe may be obtained from messenger RNA, from acomplementary strand of cDNA obtained by reverse transcription ofmessenger RNA with reverse transcriptase or by cleavage of the genome,conveniently by endonuclease digestion, followed by cloning of the geneor gene fragment in accordance with known techniques. See, for example,Kornberg, DNA Replication, W. H. Freeman and Co., San Francisco, 1980,pp. 670-679; So et al., Infect. Immun., 21:405-411, 1978. Afterisolation and characterization of the desired gene or DNA fragment, thegene or DNA fragment may be used for preparation of the probe. The probemay also be chemically synthesized using an automated synthesizer suchas a MILLIGEN® synthesizer. Chemical synthesis of the probes is thepreferred method to obtain the desired probe for use in the presentinvention.

For the most part, the oligonucleotide probe will be labeled with adetectable marker using an atom, an inorganic radical, radionucleotide,heavy metal, antibody, enzyme and the like. Conveniently, a radioactivelabel may be employed. Radioactive labels include ³² P, ³ H, ¹⁴ C andthe like. Any radioactive label may be employed which provides for anadequate signal and has sufficient half-life. Other labels includeligands, which can serve as a specific binding pair member to a labeledantibody, fluorescers, chemiluminescers, enzymes, antibodies which canserve as a specific binding pair member for a labeled ligand and thelike. A wide variety of labels have been employed in immunoassays whichcan be readily employed in the present assay. The choice of label willbe governed by the effect of the label on the rate of hybridization andbinding of the probe to the genetic DNA. It will be necessary that thelabel provide sufficient sensitivity to detect the amount of DNAavailable for hybridization. Other considerations include ease ofsynthesis of the probe, readily available instrumentation, the abilityto automate, convenience and the like.

The manner in which the label is bound to the probe will vary dependingupon the nature of the label. For a radioactive label, a wide variety oftechniques can be employed. Commonly employed is end labeling with anα-³² P-NTP and T4 polynucleotide kinase. Alternatively, nucleotides canbe synthesized where one or more of the elements present are replacedwith a radioactive isotope, e.g., hydrogen with tritium.

Where other radionucleotide labels are involved, various linking groupscan be employed. A terminal hydroxyl can be esterified with inorganicacids. For example, ³² P phosphate or ¹⁴ C organic acids can beesterified via the terminal hydroxy or esterified to provide linkinggroups to the label. Alternatively, intermediate bases may besubstituted with activatable linking groups which can be linked to thelabel.

Ligands and antiligands may be varied widely. Where a ligand has anatural receptor such as biotin, thyroxine and cortisol, the ligand canbe used in conjunction with labelled naturally occurring receptors. Anycompound can be used, either haptenic or antigenic, in combination withan antibody.

Enzymes of interest as labels will primarily be hydrolases, particularlyesterases and glycosidases, phosphatases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferoneand the like. Chemiluminescers include luciferin and2,3-dihydrophthalazinediones, i.e., luminol.

Yet another method for labeling and detecting the nucleotide probe isdisclosed in U.S. Pat. No. 4,868,103, in which an energy transferresults in the generation of bathochromic and/or delayed fluorescenceemission. Fluorescence radiation, emitted from a first energy emitter(E₁) is absorbed by a second energy emitter (E₂). The second emitteremits fluorescence radiation of a longer wavelength than the firstenergy emitter. (E₁) and (E₂) must be within a proximate distance ofeach other so that the energy emitted by (E₁) can be absorbed by (E₂)and (E₂) emits fluorescent energy at a longer wavelength.

Any means for labeling and detecting the labeled probe can be used inthe present invention. It is preferable, however, that the label isbound at a distance away from the scissile linkage and is situated atthe 3 prime or 5 prime end of the first probe oligonucleotide.

Besides having a label on the first oligonucleotide the firstoligonucleotide must also contain a scissile linkage that iscomplementary to the target molecule of interest. The scissile linkagemust be readily cleavable by restriction enzymes or other means afterhybridization has occurred. When the first oligonucleotide is a DNAsequence, the linkage may be cleaved by restriction enzymes.

The restriction enzymes are well known in the art and have specificrecognition sites. There are more than 400 different restrictionendonucleases which can be isolated from bacteria and the presentinvention contemplates the use of any of these enzymes. The criteria forchoosing the restriction enzyme is based upon the recognition site ofthe target nucleotide molecule, the first oligonucleotide probe, and theproperties of the enzyme. The first oligonucleotide probe containing thescissile linkage must be complementary to the target nucleotide'srecognition or scissile linkage site which upon hybridization of theseoligonucleotides and addition of the restriction enzyme, the duplex willbe cleaved.

Restriction enzymes or endonucleases are relatively stable proteins.Their purification to homogeneity is often not necessary. Allrestriction enzymes require a cofactor for cleavage such as Mg²⁺ or Mn²⁺and are most active in the pH range of 7.2 to 7.6. Typically for enzymecleavage an appropriate buffer system is used. These buffer systems varyamong the restriction enzymes used to cleave a hybridized duplex.Therefore, in addition to the DNA substrate and restriction enzyme, mostreaction solutions will contain TRIS buffertris(hydroxymethyl)aminomethane, Mg²⁺, NaCl, 2-mercaptoethanol andbovine serum albumin (BSA). The predominant difference among therestriction enzymes is their dependence on ionic strength. To maximizecleavage efficiencies the buffering systems are varied among therestriction enzymes. For instance, if a Hind III restriction enzyme isused in the assay, a buffer containing 50 mM NaCl, 25 mM TRIS-HCl, pH7.7, 10 mM MgCl₂, 10 mM β-mercaptoethanol and 100 μg/ml BSA is used tomaximize the cleavage efficiency. If an Eco RI enzyme is used thebuffering system contains 50 mM NaCl, 100 mM TRIS-HCl, pH 7.5, 5 mMMgCl₂ and 100 μg/ml BSA. Therefore, the present invention contemplatesthe use of different buffering systems, which vary according to therestriction enzyme used in the assay.

Restriction enzymes may also vary in temperature optima. Most cleavagesare performed at 37° C., but a few endonucleases such as Sma I preferlower incubation temperatures, and several, mainly those isolated fromthermophiles such as Taq I, require much higher temperatures. Therefore,the reaction temperature in the present invention is chosen taking intoconsideration the restriction enzyme used in the assay.

Besides cleaving the double-stranded molecule formed after hybridizationwith restriction enzymes, any other method available can be used in thecleavage process. For instance, certain chemicals may be used to cleavedouble-stranded complexes at specific sites.

The present invention also uses a second oligonucleotide whichreconstitutes with the cleaved target molecule of interest such thathybridization with the probe oligonucleotide occurs within the assay.The second oligonucleotide acts to recycle the target sequence ofinterest and thereby amplifies the amount of cleaved probe for detectionpurposes. This second oligonucleotide is a single-stranded nucleotidethat is complementary to an end of the first probe oligonucleotide suchas the 3 prime end or 5 prime end. The second oligonucleotide may bederived via processes similar to the synthesis of the firstoligonucleotide, as described above.

Besides being complementary to the 3 prime or 5 prime end of the firstoligonucleotide, the second oligonucleotide should contain bases such asa 5 prime base or 3 prime base, that will reconstitute the targetmolecule at the site that was cleaved by the restriction enzyme. Afterreconstituting with the cleaved end of the target molecule,hybridization once again occurs with the first labeled oligonucleotideand the process "cycles" once again and releases the labeled probe intothe reaction media. The second oligonucleotide may contain up to 100bases, preferably between about 10 to 20 bases, more preferably about 14bases. FIGS. 1 and 2 are illustrative examples of how the presentinvention works.

The assay is initiated by denaturing the sample target molecule to forma single-stranded molecule. The denaturation of the target molecule orsubstrate is generally performed by boiling. In addition, the substrate,first oligonucleotide, second oligonucleotide buffer and distilled watermay be mixed together and boiled for 5 to 10 minutes. The mixture isthen allowed to cool to room temperature. After cooling, the restrictionenzyme may then be added to initiate the reaction.

The temperature at which the assay is run may vary according to therestriction enzyme used, the length of the oligonucleotide probe and theG+C content of the oligonucleotides present in the assay. Suggs et al,in "Developmental Biology Using Purified Genes," (D. D. Brown, ed.),p.683. Academic Press, New York, 1981., developed an empirical formulato determine the appropriate hybridization temperature based on thetemperature at which an oligonucleotide DNA complex is dissociated. Theformula derived was: T_(d) =2° (number of A+T residues)+4°(number of G+Cresidues). This empirical formula can be used to estimate the reactiontemperature, but one must also take into account the restriction enzymeused in the assay. As discussed above, many restriction enzymes areactive at 37° C., but others may require higher or lower temperatures.The temperature of the reaction is chosen such that optimal rates ofhybridization, as well as cleavage of the restriction enzyme occurs.

The reaction time may vary depending upon the concentration of thesequence of interest, the stringency, the length of the complementaryfirst oligonucleotide probe sequence, the restriction enzyme used andthe like. Enough time should be provided to permit amplification of theprobe by recycling the target sequence of interest. Usually the assay isrun from 1 to 3 hours, more preferably for two hours.

After the reaction has run, it is stopped by placing an aliquot of thereaction mixture into a polyacrylamide gel loading buffer. The sample isthen electrophoresed and an autoradiograph is taken of theelectrophoresed gel. One can then proceed to quantitate the amount ofprobe generated on a scanning densitometer, if desired.

In order to further illustrate the present invention and the advantagesthereof, the following specific examples are given it being understoodthat same are intended as illustrative and in nowise limitative.

Synthesis of Oligonucleotides

Two oligonucleotides are required for the restriction amplificationprocess. The synthesis of the first oligonucleotide having the sequence⁵ 'ACC ATG GCT GAT CCT GCA GGT ACC AAT G³ ' (28 mer) and the secondoligonucleotide having the sequence ⁵ 'GGA TCA GCC ATG GT³ ' (14 mer)were prepared by phosphoramidite chemistry and were subsequentlypurified by anion exchange HPLC. The oligonucleotides synthesized by thephosphoramidite approach contained a free 5'-OH.

Labeling of First Oligonucleotide

The first oligonucleotide contained a 5'-OH group which was labeled bytransfer of the [³² P]phosphate from [γ³² P]ATP using polynucleotidekinase. The labeling reaction was carried out by dissolving eacholigonucleotide in distilled water. The concentration of eacholigonucleotide dissolved in the water was 0.5×10⁻¹¹ molar. The reactionwas performed using 1 μl of the first oligonucleotide (0.5×10⁻¹¹ molar),5 μl of kinase buffer which contained 0.5 M TRIS-HCl at pH 7.6, 1 mMspermidine, 50 mM dithiothreitol, 1 mM EDTA and 0.1 M MgCl₂, 30.5 mldistilled water, 12.5 μl ³² P-ATP (0.125 millicuries or 3000curies/μmole) and 1 μl of polynucleotide kinase (10 units). Theabove-mentioned reaction ingredients were combined and incubated at 37°C. for 1 hour. The reaction was terminated by heating the reactedmixture to 80° C. for 10 minutes.

The labeled first oligonucleotide prepared by this method is stable forat least one week when stored at -20° C.

HPV 16 Substrate

The HPV16-DNA virus was isolated from a cervical lesion by methods knownin the art and cloned into pBR322 using standard techniques. The clonedDNA was isolated by a modified procedure described by H. C. Birnboim andJ. Daly, Nucleic Acids Res., 7, 1513 (1979).

The cells were grown overnight in 5 ml of LB broth containing 100 μg/mlampicillin at 37° C. with vigorous shaking. 1.5 ml of the culture wastransferred to a 1.5 ml centrifuge tube and centrifuged at 10,000 g for1 minute. The supernatant was then carefully removed leaving the pelletas dry as possible. The cells were resuspended by vortexing in 100 μl ofan ice-cold solution containing 50 mM glucose, 10 mM EDTA and 25 mMTRIS-HCl at pH 8.0. The resuspended cells were allowed to incubate for 5minutes at room temperature. 20 μl of a freshly prepared solutioncontaining 0.2 N NaOH, 1% sodium dodecyl sulfate (SDS) was added to thecells and mixed by inversion. The cells were further incubated for 5minutes at 0° C.

After incubation, 150 μl of ice-cold potassium acetate at pH 4-8(prepared by adding 11.5 ml glacial acetic acid and 28.5 ml of water to60 ml of 5 M potassium acetate) was added and the mixture was invertedfor 10 seconds and incubated at 0° C. for 5 minutes.

The mixture was then centrifuged for 5 minutes at 10,000 g, and thesupernatant was transferred to another tube. The supernatant wascentrifuged again at 10,000 g for 5 minutes, and the supernatant wastransferred to another fresh test tube. RNase A was then added to thesupernatant, having a final concentration of 20 μg/ml. The reaction wasincubated at 37° C. for 20 minutes.

After incubation, an equal volume of phenol: chloroform (1:1, saturatedwith 50 mM TRIS-HCl at pH 8.0, 100 mM NaCl, 1 mM EDTA) was added and themixture was vortexes for 30 seconds and centrifuged at 10,000 g for 30seconds. The aqueous phase was then transferred to a fresh test tube.

2.5 volumes of ethanol was then added to the aqueous phase and mixed.The mixture was incubated at -70° C. for 5 minutes. The mixture wascentrifuged at 10,000 g for 5 minutes and the supernatant was removed.The pellet was then rinsed by adding 1 ml of prechilled 70% ethanol andmixed briefly. The mixture was then centrifuged for 1 minute. Thesupernatant was then removed, and the pellet was dried under vacuum.

The DNA obtained from this procedure was dissolved in 20 μl of deionizedwater.

Restriction Amplification Assay

The restriction amplification assay was initiated by combining the HPV16 substrate, the first oligonucleotide labeled probe, the firstunlabeled oligonucleotide, a PST I buffer, and deionized water. In thisRAMP assay two controls were simultaneously run with the samples. Twodifferent temperatures of 32° C. and 37° C. were utilized in thisexample.

Stock solutions of the reaction components were first prepared. Theoligonucleotides were diluted to 0.5×10⁻¹¹ molar solutions. Similarly,the second oligonucleotide was also diluted to create a 0.5×10⁻¹¹ stocksolution. The HPV16 stock was diluted with distilled water to form a 100μg/ml stock solution. The Pst I restriction enzyme stock solutioncontained 50 units/μl.

A specific aliquot was taken from these stock solutions for each assay.The total reaction volume was 50 μl. Each reaction assay contained 1 μl(100 μg) of HPV16, 2 μl of the labeled first oligonucleotide, 2 μl ofunlabeled first oligonucleotide, 4 μl of the second oligonucleotide, 5μl of Pst I buffer containing 100 mM NaCl, 10 mM TRIS-HCl at pH 7.7, 10mM MgCl₂, 1 mM DTT and 100 μg/ml BSA. 5 μl of Pst I was used. Distilledwater was added in varying quantities such that the final volume in eachassay tube was equal to 50 μl.

Five assays were run in separate tubes. The first tube (#1) contained 2μl of the first oligonucleotide labeled with ³² P, 2 μl of coldunlabeled first oligonucleotide, 4 μl of the second oligonucleotide, 5μl Pst I buffer and 32 μl distilled water. No HPV-16 was added to thefirst assay mixture.

The second assay tube (#2) contained 1 μl (100 μg) HPV-16, 2 μl of thefirst oligonucleotide labeled with ³² P, 2 μl of cold unlabeled firstnucleotide, 4 μl of the second oligonucleotide, 5 μl of Pst I buffer and31 μl of distilled water.

The third assay tube (#3) contained 1 μl of HPV-16 (100 μg), 2 μl of thefirst ³² P oligonucleotide, 2 μl of cold first oligonucleotide, 5 ml ofPst I buffer, and 35 μl of distilled water.

The fourth assay tube (#4) contained 1 μl of HPV-16 (100 μg), 2 μl ofthe first ³² P oligonucleotide, 2 μl of cold unlabelled firstoligonucleotide, 5 μl of Pst I buffer, and 35 μl of distilled water.

The fifth assay tube (#5) contained 1 μl of HPV-16 (100 μg), 2 μl oflabelled ³² P first oligonucleotide, 2 μl of cold unlabelled firstoligonucleotide, 4 μl of second oligonucleotide, 5 μl of Pst I buffer,and 31 μl of distilled water.

Prior to addition of Pst I, the five assay tubes described above wereboiled for 5 to 10 minutes and then cooled at room temperature forapproximately 10 minutes.

After cooling the samples, the samples were placed in an incubator;assay tube numbers 1, 4, and 5 discussed above were incubated at 37° C.Assay tubes numbers 2 and 3 were incubated at 32° C. To initiate theassay, 5 μl of Pst I enzyme was added to each assay tube. The assay wasrun for 2 hours.

After incubation, the reaction was stopped by adding 10 μl of a solutioncontaining 80% formamide, 15 mM TRIS-HCl at pH 7.6, 1 mM EDTA, 0.1% w/vbromophenol blue and 0.1% w/v xylene cyanole FF to each assay tube. Eachtube was then heated for 5 minutes at 65° C. and cooled rapidly.

Polyacrylamide gels were prepared by making a stock solution containing30% acrylamide (19:1 acrylamide:bis), 19 grams of acrylamide, 1 gram ofN,N'-methylenebisacrylamide and enough deionized water to dilute thesolution to 67 ml total volume. A concentrated solution of TBE bufferwas prepared by diluting to 1 liter, 108 grams of TRIS base, 55 grams ofboric acid and 9.3 grams of Disodium EDTA·2H₂ O. The pH of theconcentrated TBE buffer should be adjusted to 8.3, if appropriate. 48grams of ultra-pure grade urea (8 M) was added to the stock solutioncontaining the 30% acrylamide. 10 ml of TBE buffer was added and theurea was dissolved in this solution. 50 μl (per 100 ml of acrylamidesolution) of N,N,N',N'-tetramethylethylenediamine(TEMED) and 1 ml (per100 ml) of 10% ammonium persulfate was added and the solution was mixedwell. The gel was poured between two glass plates into theelectrophoresis apparatus and the comb was inserted immediately.

20 μl of each sample was loaded into the polymerized gel and a runningbuffer was added that contained 0.089 M TRIS-borate, pH 8.3 and 0.025 MDisodium EDTA. The samples were electrophoresed at 80 volts for 4 hours.

The gels were then wrapped in a plastic folder placed next to KodakX-OMAT AR film and exposed for 10 minutes.

FIG. 3 is an illustrative example of the autoradiograph of the presentinvention. From the autoradiograph, one can easily determine theformation of the substrate by the presence of the 28 mer oligonucleotideband and the 14 mer oligonucleotide band. Lanes 2 and 5 illustrate adoublet pattern when the substrate is present in the reaction media attwo different temperatures. Lanes 3 and 4 are indicative of the presenceof only the labeled first oligonucleotides without the secondoligonucleotide used to recycle the substrate. Lane 1 is indicative ofthe pattern obtained when both of the first and second oligonucleotidesare present in the assay mixture, but no substrate (i.e., HPV-16) waspresent in the reaction.

While the invention has been described in terms of various preferredembodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions, and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the present invention be limited solely by the scope of thefollowing claims, including equivalents thereof.

What is claimed is:
 1. A method for detecting the presence of a nucleic acid sequence which contains a scissile linkage that is cleavable by a cleaving enzyme in a biological sample, said method comprising the steps of:(a) providing a first oligonucleotide which comprises a nucleic acid sequence and a scissile linkage and is substantially complementary to a nucleic acid target sequence, said first oligonucleotide having a detectable marker attached thereto; (b) adding to form a mixture a second oligonucleotide that is substantially complementary to an end of the first oligonucleotide and comprises at least one base that will reconstitute the cleaved scissile linkage on a target molecule; (c) adding a cleaving enzyme to said reaction mixture which is able to cleave the scissile linkage if said target sequence and said first oligonucleotide hybridize; (d) hybridizing said reaction mixture; and (e) detecting the cleaved detectable marker in the presence of the first uncleaved oligonucleotide having a detectable marker attached thereto.
 2. The method according to claim 1, wherein said target molecule comprises a single-stranded DNA sequence.
 3. The method according to claim 2, wherein said first oligonucleotide comprises a single-stranded DNA sequence.
 4. The method according to claim 2, wherein said second oligonucleotide comprises a single-stranded DNA sequence.
 5. The method according to claim 1, wherein said cleaving enzyme is a restriction enzyme.
 6. The method according to claim 1, wherein said target molecule is single-stranded or double-stranded.
 7. The method according to claim 1, wherein said hybridization is carried out at a temperature to enhance the efficiency of said cleaving enzyme and the hybridization of the oligonucleotides.
 8. The method according to claim 7, wherein said hybridization is carried out at 37° C.
 9. The method according to claim 7, wherein said hybridization is carried out at 32° C.
 10. The method according to claim 7, wherein said hybridization is carried out between 1 to 4 hours.
 11. The method according to claim 10, wherein said hybridization is carried out for about 2 hours.
 12. The method according to claim 1, wherein said reaction mixture further comprises a buffering system.
 13. The method according to claim 12, wherein said buffering system comprises a MgCl₂, NaCl, tris(hydroxymethyl)aminomethane-hydrochloride (TRIS-HCl), dithiothreitol (DTT) and bovine standard albumin (BSA).
 14. The method according to claim 13, wherein said buffering system comprises 100 mM NaCl, 10 mM tris(hydroxymethyl)aminomethane-hydrochloride (TRIS-HCl), pH7.7, 10 mM MgCl₂, 1 mM DTT and 100 μg/ml BSA.
 15. The method according to claim 1, wherein said detectable marker in said first oligonucleotide is a radioactive marker.
 16. The method according to claim 15, wherein said radioactive marker is selected from the group consisting of ³² p, ³ H, ¹⁴ C and ³⁵ S.
 17. The method according to claim 16, wherein said radioactive marker is ³² P.
 18. The method according to claim 1, wherein said detectable marker in said first oligonucleotide is an enzyme marker.
 19. The method according to claim 1, wherein said detectable marker in said first oligonucleotide is a ligand which can serve as a specific binding pair member to a labeled compound selected from the group consisting of an antibody, fluorescer, chemiluminescer, enzymes, biotin and mixtures thereof.
 20. The method according to claim 1, wherein said first oligonucleotide is up to 100 mer.
 21. The method according to claim 20, wherein said first oligonucleotide is between about 10 mer to 40 mer.
 22. The method according to claim 21, wherein said first oligonucleotide is about 28 mer.
 23. The method according to claim 1, wherein said second oligonucleotide is up to 100 mer.
 24. The method according to claim 23, wherein said second oligonucleotide is between about 10 mer to 20 mer.
 25. The method according to claim 24, wherein said second oligonucleotide is about 14 mer.
 26. The method according to claim 1, further comprising the step of adding to said hybridization reaction mixture a terminating solution.
 27. The method according to claim 26, wherein said terminating solution comprises 80% formamide, 15 mM tris(hydroxymethyl)aminomethane-hydrochloride (TRIS-HCl), pH 7.6, 1 mM EDTA, 0.1% w/v bromophenol blue and 0.1% w/v xylene cyanole FF.
 28. The method according to claim 27, wherein 100 μl of said terminating solution is added to said reaction mixture.
 29. The method according to claim 2, wherein said nucleic acid target sequence is HPV
 16. 30. The method according to claim 1, Wherein said detecting step (e) comprises electrophoresing said reaction mixture and autoradiographing said electrophoresed reaction mixture.
 31. A method of detecting in a sample the presence of a nucleic acid sequence which contains a scissile linkage that is cleavable by a cleaving enzyme, said method comprising the steps of:(a) forming a reaction mixture by denaturing a target nucleic acid sequence containing a scissile linkage in the presence of: (i) a first oligonucleotide which contains a scissile linkage and is substantially complementary to the nucleic acid sequence of the target molecule; (ii) a second oligonucleotide that is substantially complementary to an end of said first oligonucleotide and comprises a 5 prime base or bases that will reconstitute the cleaved scissile linkage on said target molecule; (b) adding a cleaving enzyme to said denatured mixture; (c) permitting said reaction mixture to hybridize whereby the cleaving enzyme will release the detectable marker from said first oligonucleotide sequence; and (d) detecting the cleaved detectable marker.
 32. The method according to claim 31, wherein said target molecule comprises a single-stranded or double-stranded DNA sequence.
 33. The method according to claim 32, wherein said first oligonucleotide comprises a single-stranded DNA sequence.
 34. The method according to claim 32, wherein said second oligonucleotide comprises a single-stranded DNA sequence.
 35. The method according to claim 31, wherein said cleaving enzyme is a restriction enzyme.
 36. The method according to claim 31, wherein said target molecule is single-stranded.
 37. The method according to claim 31, wherein said hybridization is carried out at a temperature to enhance the efficiency of said cleaving enzyme and the hybridization of the oligonucleotides.
 38. The method according to claim 37, wherein said hybridization is carried out at 37° C.
 39. The method according to claim 37, wherein said hybridization is carried out at 32° C.
 40. The method according to claim 37, wherein said hybridization is carried out between 1 to 4 hours.
 41. The method according to claim 40, wherein said hybridization is carried out for about 2 hours.
 42. The method according to claim 31, wherein said reaction mixture further comprises a buffering system.
 43. The method according to claim 42, wherein said buffering system comprises a MgCl₂, NaCl, tris(hydroxymethyl)aminomethane-hydrochloride (TRIS-HCl), dithiothreitol (DTT) and bovine standard albumin (BSA).
 44. The method according to claim 43, wherein said buffering system comprises 100 mM NaCl, 10 mM tris(hydroxymethyl)aminomethane-hydrochloride (TRIS-HCl), pH7.7, 10 mM MgCl₂, 1 mM DTT and 100 μg/ml BSA.
 45. The method according to claim 31, wherein said detectable marker in said first oligonucleotide is a radioactive marker.
 46. The method according to claim 45, wherein said radioactive marker is selected from the group consisting of ³² P, ³ H, ¹⁴ C and ³⁵ S.
 47. The method according to claim 46, wherein said radioactive marker is ³² P.
 48. The method according to claim 31, wherein said detectable marker in said first oligonucleotide is an enzyme marker.
 49. The method according to claim 31, wherein said detectable marker in said first oligonucleotide is a ligand which can serve as a specific binding pair member to a labeled compound selected from the group consisting of an antibody, fluorescer, chemiluminescer, enzymes, biotin and mixtures thereof.
 50. The method according to claim 31, wherein said first oligonucleotide is up to 100 mer.
 51. The method according to claim 50, wherein said first oligonucleotide is between about 10 mer to 40 mer.
 52. The method according to claim 51, wherein said first oligonucleotide is about 28 mer.
 53. The method according to claim 31, wherein said second oligonucleotide is up to 100 mer.
 54. The method according to claim 53, wherein said second oligonucleotide is between about 10 mer to 20 mer.
 55. The method according to claim 54, wherein said second oligonucleotide is about 14 mer.
 56. The method according to claim 31, further comprising the step of adding to said hybridization reaction mixture a terminating solution.
 57. The method according to claim 56, wherein said terminating solution comprises 80% formamide, 15 mM tris(hydroxymethyl)aminomethane (TRIS-HCl), pH 7.6, 1 mM EDTA, 0.1% w/v bromophenol blue and 0.1% w/v xylene cyanole FF.
 58. The method according to claim 56, wherein 100 μl of said terminating solution is added to said reaction mixture.
 59. The method according to claim 32, wherein said nucleic acid target sequence is HPV
 16. 60. The method according to claim 31, wherein said detecting step (e) comprises electrophoresing said reaction mixture and autoradiographing said electrophoresed reaction mixture.
 61. A method for detecting the presence of a nucleic acid sequence which contains a scissile linkage that is cleavable by a cleaving enzyme in a biological sample, said method comprising the steps of:(a) hybridizing a target molecule having a scissile linkage to a labelled first oligonucleotide probe that has a complementary sequence to said target and a detectable marker to provide a probe:target duplex; (b) cleaving said duplex at the scissile linkage; (c) adding a second oligonucleotide that reconstitutes the cleaved scissile linkage on the target molecule; (d) recycling the reconstituted target molecule; and (e) detecting said cleaved first oligonucleotide probe.
 62. The method according to claim 61, wherein said target molecule comprises a single-stranded DNA sequence.
 63. The method according to claim 62, wherein said first oligonucleotide comprises a single-stranded DNA sequence.
 64. The method according to claim 62, wherein said second oligonucleotide comprises a single-stranded DNA sequence.
 65. The method according to claim 61, wherein said cleaving step (b) is carried out with a restriction enzyme.
 66. The method according to claim 61, wherein said target molecule is single-stranded.
 67. The method according to claim 61, wherein said hybridization is carried out at a temperature to enhance the efficiency of said cleaving enzyme and the hybridization of the oligonucleotides.
 68. The method according to claim 67, wherein said hybridization is carried out at 37° C.
 69. The method according to claim 67, wherein said hybridization is carried out at 32° C.
 70. The method according to claim 67, wherein said hybridization is carried out between 1 to 4 hours.
 71. The method according to claim 70, wherein said hybridization is carried out for about 2 hours.
 72. The method according to claim 61, wherein said cleaving step (b) is further carried out in a buffering system.
 73. The method according to claim 72, wherein said system comprises a MgCl₂, NaCl, tris(hydroxymethyl)aminomethane-hydrochloride (TRIS-HCl), dithiothreitol (DTT) and bovine standard albumin (BSA).
 74. The method according to claim 73, wherein said buffering system comprises 100 mM NaCl, 10 mM tris(hydroxymethyl)aminomethane-hydrochloride (TRIS-HCl), pH7.7, 10 mM MgCl₂, 1 mM DTT and 100 μg/ml BSA.
 75. The method according to claim 61, wherein said detectable marker in said first oligonucleotide is a radioactive marker.
 76. The method according to claim 75, wherein said radioactive marker is selected from the group consisting of ³² P, ³ H, ¹⁴ C and ³⁵ S.
 77. The method according to claim 76, wherein said radioactive marker is ³² P.
 78. The method according to claim 61, wherein said detectable marker in said first oligonucleotide is an enzyme marker.
 79. The method according to claim 61, wherein said detectable marker in said first oligonucleotide is a ligand which can serve as a specific binding pair member to a labeled compound selected from the group consisting of an antibody, fluorescer, chemiluminescer, enzymes, biotin and mixtures thereof.
 80. The method according to claim 61, wherein said first oligonucleotide is up to 100 mer.
 81. The method according to claim 80, wherein said first oligonucleotide is between about 10 mer to 40 mer.
 82. The method according to claim 81, wherein said first oligonucleotide is about 28 mer.
 83. The method according to claim 61, wherein said second oligonucleotide is up to 100 mer.
 84. The method according to claim 83, wherein said second oligonucleotide is between about 10 mer to 20 mer.
 85. The method according to claim 84, wherein said second oligonucleotide is about 14 mer.
 86. The method according to claim 61, further comprising the step of adding to said hybridization reaction mixture a terminating solution.
 87. The method according to claim 86, wherein said terminating solution comprises 80% formamide, 15 mM tris(hydroxymethyl)aminomethane-hydrochloride (TRIS-HCl), pH 7.6, 1 mM EDTA, 0.1% w/v bromophenol blue and 0.1% w/v xylene, cyanole FF.
 88. The method according to claim 87, wherein 100 μl of said terminating solution is added to said reaction mixture.
 89. The method according to claim 62, wherein said nucleic acid target sequence is HPV
 16. 90. The method according to claim 61, wherein said detecting step (e) comprises electrophoresing said reaction mixture and autoradiographing said electrophoresed reaction mixture.
 91. The method according to claim 5, wherein said restriction enzyme is selected from the group consisting of Aat II, Acc I, Acc III, Aha II, Alu I, Acc I, Apa I, ApaL I, Ava I, Val I, Bam HI, Bcl I, Bgl I, BssH II, BstE II, Cla I, Dra I, Eco52 I, Eco RI, EcoR II, EcoR V, Fsp I, Hae III, Hha I, Hind III, Hpa I, Kpn I, Ksp I, Nci I, Mst II, Nae I, Pst I, Puv I, and Xba I.
 92. The method according to claim 35, wherein said restriction enzyme is selected from the group consisting of Aat II, Acc I, Acc III, Aha II, Alu I, Aoc I, Apa I, ApaL I, Ava I, Bal I, Bam HI, Bcl I, Bgl I, BssH II, BstE II, Cla I, Dra I, Eco52 I, Eco RI, EcoR II, EcoR V, Fsp I, Hae III, Hha I, Hind III, Hpa I, Kpn I, Ksp I, Nci I, Mst II, Nae I, Pst I, Puv I, and Xba I.
 93. The method according to claim 92, wherein said restriction enzyme is Eco RI.
 94. The method according to claim 92, wherein said restriction enzyme is Pst I.
 95. The method according to claim 65, wherein said restriction enzyme is selected from the group consisting of Aat II, Acc I, Acc III, Aha II, Alu I, Acc I, Apa I, ApaL I, Ava I, Bal I, Bam HI, Bcl I, Bgl I, BssH II, BstE II, Cla I, Dra I, Eco52 I, Eco RI, EcoR II, EcoR V, Fsp I, Hae III, Hha I, Hind III, Hpa I, Kpn I, Ksp I, Nci I, Mst II, Nae I, Pst I, Puv I, and Xba I.
 96. The method according to claim 95, wherein said restriction enzyme is Eco RI.
 97. The method according to claim 95, wherein said restriction enzyme is Pst I.
 98. The method according to claim 29, wherein said first oligonucleotide has the sequence:

    .sup.5 'ACC ATG GCT GAT CCT GCA GGT ACC AAT G.sup.3 '.


99. The method according to claim 29, wherein said second oligonucleotide has the sequence:

    .sup.5 'GGA TCA GCC ATG GT.sup.3 '.


100. The method according to claim 59, wherein said first oligonucleotide has the sequence:

    .sup.5 'ACC ATG GCT GAT CCT GCA GGT ACC AAT G.sup.3 '.


101. The method according to claim 59, wherein said second oligonucleotide has the sequence:

    .sup.5 'GGA TCA GCC ATG GT.sup.3 '.


102. The method according to claim 89, wherein said first oligonucleotide has the sequence:

    .sup.5 'ACC ATG GCT GAT CCT GCA GGT ACC AAT G.sup.3 '.


103. The method according to claim 89, wherein said second oligonucleotide has the sequence:

    .sup.5 'GGA TCA GCC ATG GT.sup.3 '. 